Project title: Molecular mechanism of piRNA biogenesis. Project Summary: Non-coding RNAs have diverse functions in eukaryotic cells. Use of these non-coding RNAs in therapeutic approaches is a promising but rather unexplored direction in biomedical research. A few years ago, we discovered a new class of small non-coding RNAs, termed piwi-interacting RNAs (piRNAs), that are expressed in animal germlines. piRNAs, together with their protein partners, Piwi proteins, recognize and silence endogenous genomic parasites called transposable elements. The silencing of transposons is critical in germline cells and the failure of piRNA-mediated repression leads to sterility in both Drosophila and mice. The mechanism of biogenesis of piRNAs appears to be distinct from that of other classes of small non-coding RNAs, microRNA and siRNA. piRNAs are encoded in distinct genomic regions dubbed piRNA clusters that work as memory banks to store inactive copies of past transposon invaders. piRNA clusters produce long transcripts that are further processed to mature small RNA species. Our goal is to dissect piRNA biogenesis from their transcription to processing into mature piRNAs and to understand how these steps are regulated. We want to understand how transcripts that are destined for processing into piRNAs are selected and identify the enzymatic machinery responsible for processing. To meet these objectives we will dissect the molecular mechanism of piRNA biogenesis using D. melanogaster and insect cell lines. We will investigate the genomic and chromatin structure of piRNA clusters and map their promoter regions. These studies will identify regulatory elements necessary for piRNA expression and determine the role that chromatin plays in regulation of piRNA transcription. Next we will identify determinants in precursor transcripts that are required for piRNA biogenesis. We will determine if piRNA processing depends on distinct sequence elements inside the piRNA precursor transcripts. We will also search for a protein complex directly responsible for piRNA processing. Finally, we will study the intracellular localization of piRNA precursor transcripts and the role of nuage granules in piRNA biogenesis. Together, these experiments will comprehensively analyze all steps of piRNA biogenesis from transcription of precursor RNAs to processing into mature small RNAs. The failure of piRNA repression leads to sterility, and also might be important in aging and cancer progression. Thus, our findings will provide the basis for directing the piRNA pathway in order to make these non-coding RNAs useful tools in epigenetic research and in therapy.